The long term goal of this proposal is to develop and apply new analytical methods for extracting accurate structural information from site-directed spin labeling (SDSL) studies of proteins using EPR spectroscopy. These advances will provide a new dimension to modem structural studies. Given the rapidly growing number of SDSL for determining structural features and functionally relevant structural transitions of proteins including integral membrane proteins, the methods developed will have an immediate and general impact on modern structural biology. 0The major emphasis of the proposed work, in the short term, is the development of methods for determining accurate interprobe distances and the distribution of orientations between two interacting spin labels site- specifically incorporated into proteins. The proposed methods are logical extensions of previous work that has demonstrated the importance of multifrequency EPR and advanced global analysis tools for rigorous data interpretation. Four Specific Aims will be addressed: l) New global analysis algorithms will be developed for extracting interprobe distance and relative orientation from multifrequency EPR data for the case of partial static ordering between the probes; 2) This new algorithm will be employed to analyze experimental data from di-spin labeled helical peptides and from double SDSL mutants of T4 lysozyme. These studies will provide an important verification of the accuracy and capabilities of the methods developed; 3) This same algorithm will be employed to determine interprobe distances and relative orientations for mutants of alphaAcrystallin and HSP 27, and these constraints will then be employed to construct tertiary and quaternary structural models for the putative substrate binding domain and the intersubunit interface of the homo- oligomeric species formed by these proteins; and 4) New global analysis algorithms will be developed to extract interprobe distance and average relative orientation from multifrequency EPR data for the case where there is dynamic disordering between the probes. The general hypothesis of this project is that accurate distance and geometry information can be obtained from SDSL studies of protein structure under a wide range of experimental conditions and that by combining these complementary structural constraints, many fewer mutants will have to be characterized in order to adequately constrain tertiary structural models and to fully characterize structural transitions which involve domain movements in proteins.